The following is a discussion of relevant art pertaining to nucleic acid detection techniques. The discussion is provided only for understanding of the invention that follows. The summary is not an admission that any of the work described below is prior art to the claimed invention.
Nucleic acid molecules such as double stranded RNA (e.g., siRNA), antisense, ribozymes, DNAzymes, aptamers, decoys, 2′,5′-oligoadenylate molecules and triplex forming oligonucleotides, are becoming increasingly important therapeutic modalities for the treatment of disease. As these molecules are developed as drugs, the need arises to detect the presence of, and/or measure the concentrations of these compounds in biological samples in order to optimize drug design, and to monitor and optimize patient treatment regimens. Conventional approaches to bioanalytical quantitation of nucleic acid molecules have included HPCL analysis and electrophoresis, such as capillary gel electrophoresis. These approaches tend to have limited sensitivity (e.g., from 50 to 100 ng/mL), and are time intensive because they require extraction of the nucleic acid of interest from a biological sample or matrix. Furthermore, this extraction step can contribute to limited assay sensitivity dut to incomplete recovery of the analyte. Other detection methods for quantitating nucleic acid molecules in biological samples include the use of radio-labeled oligonucleotides. However, the use to radio-labeled nucleotides is limited with regard to pre-clinical research settings and are not suitable for use in human subjects. Alternative methods often rely upon hybridization sandwich assays that can detect single stranded oligonucleotides in a sample. For example, Ishii et al., U.S. Pat. No. 5,474,895, describes certain nucleic acid sandwich hybridization assays for detecting single stranded oligonucleotides using polystyrene solid supports. Holtke et al., U.S. Pat. No. 5,354,657, describes certain processes for the detection of single stranded nucleic acids using certain nucleic acid sandwich hybridization assays that utilize a steroid hapten detection probe. Usman et al., International PCT Publication No. WO 01/66721, describes nucleic acid sensor molecules including halfzymes for detecting and quantitating nucleic acids, polynucleotides and oligonucleotides in a sample. Seiwert et aL, International PCT Application No. PCT/US02/35529, describes nucleic acid sensor molecules including halfzymes for detecting and quantitating nucleic acids, polynucleotides and oligonucleotides in a sample. Radka et al., U.S. Ser. No. 10/366,191, describes the use of antibody mediated detection assays for detecting and quantitating nucleic acids, polynucleotides and oligonucleotides in a sample.